The present invention relates to methods of incorporating membrane proteins into preformed liposomes. It further relates to methods of making a prothrombin time (PT) reagent using purified, reconstituted natural or recombinant human tissue factor (rTF). More particularly, the invention relates to the reconstitution of tissue factor (TF) into phospholipid liposomes to produce a tissue factor-based PT reagent.
Membrane proteins are critical for cellular function and include receptors, ion pumps, electron transport proteins, signal transducers and regulators of the intracellular environment. The isolation and reconstitution of these proteins into membranes has been well studied and is well documented.
Liposomes are a general category of vesicles which comprise one or more lipid bilayers surrounding an aqueous space. Liposomes include unilamellar vesicles composed of a single membrane or lipid bilayer, and multilamellar vesicles (MLVs) composed of many concentric membranes (or lipid bilayers). Liposomes are commonly prepared from phospholipids. Due to unique characteristics of these vesicles, liposomes have been widely used as a model membrane for investigating the properties of biomembranes and for studying the functions of membrane proteins.
There are essentially four presently known mechanisms for incorporating, i.e., reconstituting, proteins into liposomes. See Rigaud, J-L., et al., xe2x80x9cLiposomes as Tools for the Reconstitution of Biological Systems,xe2x80x9d p. 71-88, in Liposomes as Tools in Basic Research and Industry, ed. Philippot, J. R. and Schuber, F., CRC Press, Boca Raton, Fla. (1995). One method involves the use of an organic solvent. However, such procedures often result in the denaturation of the proteins. A second method uses mechanical means to produce large and small unilamellar vesicles from MLVs by swelling of the dry phospholipid films in excess buffer. Such mechanical means include sonication of MLVs, forcing multilamellar lipid vesicles through a French press, or cycles of freeze-thawing or dehydration-rehydration. Drawbacks with sonication include variability and inactivation of certain proteins by sonication as well as production of small liposomes. A third process involves the direct incorporation of proteins into preformed small unilamellar liposomes, also termed spontaneous incorporation. Such methods are usually catalyzed by low cholate or lysolecithin concentrations. Problems with these methods include the wide size distribution of the proteoliposomes, heterogeneous distribution of the protein among the liposomes and presence of the non-phospholipid impurities, required for an effective protein incorporation, that would affect performance of those liposomes. The fourth and most often used method of incorporating proteins into liposomes involves the use of detergents. In such a method, the proteins and phospholipids are cosolubilized in a detergent to form micelles. The detergent is then removed, resulting in the spontaneous formation of bilayer vesicles with the protein incorporated therein. The detergent is incorporated into liposome as well as the protein and thus, these methods require removal of the detergent by methods such as dialysis, gel exclusion chromotography or adsorption on hydrophobic resins. The methods that use detergent are very slow because the detergent removal must be as complete as possible and also because a phase change that takes place during this process slows detergent removal even further. The detergent is also difficult to remove completely. Another disadvantage is that one cannot control the orientation of protein incorporated into the liposomes by using the detergent methods.
Liposomes have several properties which make them useful in various applications. The most important of these characteristics are the uniform controllable size and the surface characteristics which can control the biological fate of the liposomes. These properties make liposomes preferred carriers for drug delivery systems and the basis for reagents for assays. For example, liposomes containing tissue factor have been used as reagents for prothrombin time (PT) assays for testing coagulation of blood. In these cases, the phospholipid constituent of the liposomes is used as a substitute for platelet phospholipids, which are essential for normal hemostasis in vivo. For example, Dade Behring Inc. presently produces INNOVIN(copyright) for use in PT determinations and prothrombin time-based assays. This product is prepared from purified human tissue factor produced in E.coli combined with synthetic phospholipids (thromboplastin), calcium, buffers and stabilizers.
Coagulation of blood occurs by two pathways, the intrinsic pathway and the extrinsic pathway. In the intrinsic (endogenous or foreign contact dependent) pathway the chain of events leading to coagulation is set in motion merely by exposure of plasma to nonendothelial surfaces, such as glass in vitro or collagen fibers in basement membranes in vivo. In contrast, the extrinsic (exogenous or tissue-dependent) pathway is initiated when, as a result of outside injury to the vessel wall, tissue juice becomes mixed with components of the blood plasma.
It has been observed that the tissues of vertebrates, when added to citrated plasma and recalcified, will profoundly accelerate clotting time. This tissue constituent which has been observed to activate the coagulation protease cascades by the extrinsic pathway is commonly referred to as thromboplastin or tissue factor (TF).
Tissue factor is a membrane-associated glycoprotein which functions by forming a complex with blood coagulation factors VII and VIIa. The complexing of these factors greatly enhances the proteolytic activity of factors VII and VIIa. Functional activity of tissue factor has an absolute dependence on the presence of phospholipids. Bach, Ronald R., Initiation of Coagulation by Tissue Factor, CRC Critical Reviews in Biochemistry 1988; 23 (4): pp. 339-368. The factor VII/VIla/tissue factor complex activates a series of specific enzymes that comprise the extrinsic and common pathways of the coagulation cascades ultimately leading to the formation of thrombin, fibrin, platelet activation, and finally clot formation. Nemerson, Yale, Tissue Factor and Hemostasis, Blood 1988; 71:pp. 1-8.
Screening tests for coagulation disorders are designed to detect a significant abnormality in one or more of the clotting factors and to localize this abnormality to various steps in the coagulation pathway. Commonly used screening tests for this purpose include the activated partial thromboplastin time (APTT) and the prothrombin time (PT). Diagnostic tests such as the PT test, utilize this series of enzymatic events in vitro under controlled conditions to diagnose disfunctions in the blood coagulation system of patients. In the PT test, the time it takes for clot formation to occur, is the Prothrombin time or xe2x80x9cPT valuexe2x80x9d.
The PT test is performed by adding tissue thromboplastin with calcium to plasma. This initiates clotting by activating Factor VII which in turn activates Factor X which in the presence of Factor V, lead to the conversion of prothrombin to thrombin. The thrombin which is so produced converts fibrinogen to fibrin. PT therefore bypasses the intrinsic clotting pathway and is normal in patients with deficiencies of Factors XII, XI, IX and VIII. PIT is abnormal in patients with deficiencies of Factors VII, X, V, prothrombin or fibrinogen. Tissue thromboplastin is a phospholipid extract (from rabbit brain or lung and human brain or placenta) to which calcium has been added. It is usually provided in a lyophilized form and must be reconstituted with distilled water.
The prothrombin time (PT) test is the most commonly performed assay in the coagulation laboratory.
PT assay reagents are particularly useful in rapid screening tests to detect single or combined deficiencies of the extrinsic coagulation system indicative of hereditary and acquired coagulation disorders, liver disease or vitamin K deficiency. PT assay reagents are also used in monitoring tests for oral anticoagulant therapy and assays for specific coagulation factors.
Tissue factor is one example of a membrane protein. Membrane proteins (e.g. receptors) are composed of one or more transmembrane domains together with intracellular and extracellular domains. The activity of such proteins is frequently measured following integration of the purified protein into an artificial membrane. Tissue factor is a receptor for factor VII of the blood coagulation system and is composed of apoprotein and lipids (Pitlick, F. A. and Nemerson, Y., Binding of the protein component of tissue factor to phospholipids. Biochemistry (1970) 9 (26): 5105-13). The apoprotein is a glycosylated polypeptide of 263 amino acids. Close to the carboxy-terminal end, it possesses a hydrophobic sequence of 23 amino acids by which it is anchored in the membrane. The intracellular moiety is composed of 21 amino acids (Fisher, K. L. et al. Cloning and expression of human tissue factor cDNA. Thromb. Research (1987) 48: 89-99); Morrissey, J. H. et al. Molecular cloning of the cDNA for the tissue factor, the cellular receptor for the initiation of the coagulation protease cascade. Cell (1987) 50: 29-35). In vivo, tissue factor is present as an integral membrane protein of cells which are not in direct contact with the blood. Its physiological function as a cell-surface receptor comprises binding and activating plasma coagulation factor VII upon coming into contact with blood or plasma. This complex possesses serine protease activity and is able to activate factors IX and X and thereby trigger coagulation.
Fickenscher, K. and Zender, N. F. (U.S. Pat. No. 5,599,909) describe a process of relipidization of isolated tissue factor that does not use detergents and is achieved by acidifying and/or heating a protein/lipid mixture. This process involves mixing protein and phospholipids at sufficiently low pH values. In this process, the phospholipids are not dissolved with the aid of a detergent, but instead, are emulsified in an aqueous solution. Appropriate pH ranges are taught to be between pH 1 and 5, preferably between pH 2 to 4, particularly preferably at a pH of about 3. The relipidization can be carried out using a membrane protein which is dissolved or one which is bound to an affinity column (e.g., an immunoadsorption column containing a polyclonal or monoclonal antibody). An aqueous emulsion of phospholipids is initially mixed with buffer at acid pH. Purified membrane protein is subsequently added to this acidic emulsion, and mixed. After an incubation time between 1 and 10 minutes, the pH of the mixture can be adjusted to the desired value immediately after mixing the protein sample to achieve homogeneity. Subjecting these proteins to low pH may cause denaturation of the protein and affect removal of acid-labile groups, such as certain glycosidic linkages. Loss of such groups from proteins may affect specific binding sites present in these proteins.
Fickenscher and Zender (U.S. Pat. No. 5,599,909) also teach a second method for integrating membrane proteins into a lipid membrane. This method uses the process of heating a protein in the presence of phospholipids. As in the process involving acidification, the lipids are not dissolved with the aid of detergents but rather by heating the mixture at 80 to 95xc2x0 C. for 1 to 10 minutes. Subsequently, the mixture is cooled to room temperature within between 1 and 10 minutes and buffer is subsequently added. Following relipidization, the membrane protein is incorporated into a lipid membrane in active form. Suitable additives can be added and the liposomes subjected to further processing. If tissue factor apoprotein is relipidized using one of the processes according to the invention, its use as a therapeutic agent or diagnostic agent becomes possible. In the second case, the relipidized tissue factor can be processed to produce a reagent for determining the prothrombin time for the purposes of examining blood coagulation in plasma. However, heating at these temperatures may result in irreversible protein unfolding, denaturation and precipitation.
As mentioned above, INNOVIN(copyright) is one example of a commercial product in which a membrane protein is incorporated into liposomes. Because INNOVIN(copyright) is manufactured from recombinant human tissue factor and synthetic phospholipid, it does not contain any other clotting factors, such as prothrombin, Factor VIII and Factor X. Furthermore, INNOVIN(copyright) is from a pure source, unlike other commercially available PT reagents that contain crude tissue factor extracted from natural sources such as rabbit brain, rabbit brain/lung mixtures, human placenta or ox brain. Each of these sources has limitations. For example, rabbit brain thromboplastin shows seasonal variability, lot-to-lot variability and is dependent on reliable raw material sources. Human tissue factor may be a source of HIV or other human viral diseases and is also dependent on reliable sources. Ox brain gives normal PT values that are much longer than those which use tissue factor from other common sources. Longer PT values lead to less throughput in the clinical laboratory. Additionally, these longer times may reflect differences in the ability of ox tissue factor to bind human factor VII. Moreover, crude tissue factor preparations from natural sources contain other coagulation factors as contaminants. Contamination with coagulation factors results in coagulation factor assay curves that are less sensitive to coagulation factor-deficient plasmas. Therefore, it is desirable to use a source of tissue factor, which does not suffer from these drawbacks and has improved lot-to-lot variability to create a more reproducible PT reagent.
INNOVIN(copyright) is highly sensitive to factor deficiencies and oral anti-coagulant-treated plasma samples. The sensitivity of INNOVIN(copyright) is similar to the WHO human brain reference thromboplastin. INNOVIN(copyright) is also insensitive to therapeutic levels of heparin. This combination of properties makes INNOVIN(copyright) very useful for monitoring oral anticoagulation therapy. Because INNOVIN(copyright) is so sensitive, it allows differentiation of abnormal plasmas, even in mildly pathological ranges. This tissue factor reagent and the methods for preparing the same are described by Hawkins P. L., et al. (Patent No. WO 93/07492 and U.S. Pat. No. 5,625,036), both incorporated herein by reference. The reagent is generally made by combining purified tissue factor, in a detergent such as octylglucoside, with natural or synthetic phospholipids, also solubilized in a detergent solution. The detergents are then removed by diafiltration or dialysis to form lipid micelles that contain tissue factor. It would be useful to have a method of preparing a tissue factor reagent, such as INNOVIN(copyright), without the need to use dialysis or diafiltration to remove the detergent.
The present invention relates to methods for reconstituting purified membrane proteins into preformed liposomes, in the presence of at least one type of fatty acid. In one preferred embodiment, the present invention relates to such methods that do not use detergent.
The present application relates to a method of making liposomes having membrane proteins incorporated therein, the method comprising: providing the membrane protein in solution; providing a solution of preformed liposomes; and incubating the mixture under physiological conditions of temperature and pH. Prior to the step of providing a solution of preformed liposomes, the liposomes are formed by combining a mixture of phospholipids with a solution of at least one type of unsaturated fatty acid. The fatty acid comprises an unsaturated fatty acid having from about 16 to about 20 carbon atoms. Preferably, the fatty acid comprises oleic acid. In preferred methods, the oleic acid is present in an amount ranging from about 10 to about 35 weight percent, preferably from about 15 to about 30 weight percent, and most preferably about 16 weight percent.
In the method of the present invention, the membrane protein is solubilized. Preferably the protein is solubilized in a salt solution. In a preferred embodiment the protein is solubilized in 50 mM Hepes-150 mM NaCl, pH 7.40 containing 40-50% trifluoroethanol, 60% alcohol or 20-40% DMSO or by exchanging with 50 mM Hepesxe2x88x920.5 M NaCl, pH 7.40. Preferably, the protein is solubilized by exchanging a detergent solution of the protein with a buffer containing salt such as 50 mM Hepes-0.5 M NaCl, pH 7.40.
The preformed liposomes for use in the methods of the present invention comprise either natural or synthetic phospholipids. The mixture of phospholipids comprises a mixture of phospholipids containing saturated or unsaturated mono or disubstituted fatty acids and a combination thereof. These phospholipids are selected from dioleoylphosphatidylcholine, dioleoylphosphatidylserine, dioleoylphosphatidylethanolamine, dioleoylphosphatidylglycerol, dioleoylphosphatidic acid, palmitoyloleoylphosphatidylcholine, palmitoyloleoylphosphatidylserine, palmitoyloleoylphosphatidylethanolamine, palmitoyloleoylphophatidylglycerol, palmitoyloleoylphosphatidic acid, palmitelaidoyloleoylphosphatidylcholine, palmitelaidoyloleoylphosphatidylserine, palmitelaidoyloleoylphosphatidylethanolamine, palmitelaidoyloleoylphosphatidylglycerol, palmitelaidoyloleoylphosphatidic acid, myristoleoyloleoylphosphatidylcholine, myristoleoyloleoylphosphatidylserine, myristoleoyloleoylphosphatidylethanoamine, myristoleoyloleoylphosphatidylglycerol, myristoleoyloleoylphosphatidic acid, dilinoleoylphosphatidylcholine, dilinoleoylphosphatidylserine, dilinoleoylphosphatidylethanolamine, dilinoleoylphosphatidylglycerol, dilinoleoylphosphatidic acid, palmiticlinoleoylphosphatidylcholine, palmiticlinoleoylphosphatidylserine, palmiticlinoleoylphosphatidylethanolamine, palmiticlinoleoylphosphatidylglycerol, palmiticlinoleoylphosphatidic acid. These phospholipids may also be the monoacylated derivatives of phosphatidylcholine (lysophophatidylidylcholine), phosphatidylserine (lysophosphatidylserine), phosphatidylethanolamine (lysophosphatidylethanolamine), phophatidylglycerol (lysophosphatidylglycerol) and phosphatidic acid (lysophosphatidic acid). The monoacyl chain in these lysophosphatidyl derivatives may be palimtoyl, oleoyl, palmitoleoyl, linoleoyl myristoyl or myristoleoyl. Preferably, the mixture of phospholipids comprises dioleoylphosphatidylcholine and dioleoylphosphatidylserine in a ratio of from about 4 to about 1. In preferred methods, the dioleoylphosphatidylcholine and dioleoylphosphatidylserine are in a ratio of from about 7 to about 3. In preferred methods the phospholipids are synthetic.
As mentioned above, the method of the present invention involves the step of incubating the mixture of membrane protein and liposomes between 25xc2x0 and 45xc2x0 C. but preferably at about 37xc2x0 C. Preferably the mixture is heated from about 30 minutes to at least an hour, preferably for about an hour.
The mixture is then diluted with the appropriate buffer, for example, Hepes-based buffer, pH 7.4 containing salt, BSA, calcium chloride, dextran, glycine and preservative.
The methods of the present invention further relate to the method of making a reagent comprising tissue factor reconstituted into preformed liposomes. The method of the present invention for making a tissue factor reagent comprises: providing tissue factor in solution; providing a solution of preformed liposomes comprising a mixture of phospholipids and at least one type of unsaturated fatty acid; and incubating the mixture under the physiological temperature of 37xc2x0 C. The mixture of phospholipids preferably comprises a mixture of phospholipids selected from dioleoylphosphatidylcholine, dioleoylphosphatidylserine dioleoylphosphatidylethanolamine, dioleoylphosphatidylglycerol, dioleoylphosphatidic acid and the fatty acid preferably comprises oleic acid.
The methods of the present invention provide a method to combine membrane proteins, e.g., tissue factor, with preformed liposomes which is more efficient and reproducible than presently used methods. The present invention also relates to a PT reagent which has a high degree of sensitivity and reproducibility for determining PT values. The PT reagent of the present invention is sensitive to the overall function of the coagulation system. The methods of the present invention provide a PT reagent with a well-defined clotting time for normal plasma samples and which prolongs the clotting times of abnormal plasma samples. The methods of the present invention also provide a PT reagent with minimal lot-to-lot variability and enhanced stability and optical clarity.